US3953417A - Synthetically modified trypsin inhibitors and process for preparing them - Google Patents

Synthetically modified trypsin inhibitors and process for preparing them Download PDF

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Publication number
US3953417A
US3953417A US05/503,066 US50306674A US3953417A US 3953417 A US3953417 A US 3953417A US 50306674 A US50306674 A US 50306674A US 3953417 A US3953417 A US 3953417A
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US
United States
Prior art keywords
obu
glu
inhibitor
trypsin
callicrein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
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US05/503,066
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English (en)
Inventor
Wolfgang Konig
Oswald Zwisler
Gerhard Guthorlein
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Hoechst AG
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Hoechst AG
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Filing date
Publication date
Priority claimed from DE19732344886 external-priority patent/DE2344886C3/de
Application filed by Hoechst AG filed Critical Hoechst AG
Priority to US05/636,728 priority Critical patent/US3992529A/en
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Publication of US3953417A publication Critical patent/US3953417A/en
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Expired - Lifetime legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S930/00—Peptide or protein sequence
    • Y10S930/01—Peptide or protein sequence
    • Y10S930/29—Polyamino acid or polypeptide with an uninterrupted series of peptide repeating units

Definitions

  • This trypsin-callicrein inhibitor is a basic polypeptide with a molecular weight of 6500 and an isoelectric point of about 10.5. It consists of a single peptide chain of 58 amino-acids, the primary structure of which is known from Biochemical and Biophysical Research Communications Vol. 20, pages 463 to 468 (1965) and which is cross-linked by 3 disulfide bridges.
  • this inhibitor inhibits various proteinases and esterases, for example trypsin, chymotrypsin, cathepsin, plasmin and callicrein.
  • various proteinases and esterases for example trypsin, chymotrypsin, cathepsin, plasmin and callicrein.
  • trypsin trypsin
  • chymotrypsin cathepsin
  • plasmin plasmin
  • callicrein callicrein.
  • the inhibitor is stored after a relatively short time, preferably in the kidneys of the test animal or of the human. It is assumed today that this is because the strongly basic inhibitor molecule is bound in unspecific manner to acid muco-polysaccharides or nucleic acids.
  • the activity of the inhibitor is only slightly modified or not at all by prolongation of the 5 present carboxyl groups with acid peptides, but that because of the lowering of the isoelectric point the modified inhibitor is excreted much faster from the kidney than an unmodified molecule.
  • the present invention provides semi-synthetically prepared derivatives of the basic trypsin-callicrein inhibitor from organs of mammals, in which the 5 carboxyl groups present in the peptide molecule are amidated by peptide groups of the general formula I ##EQU2## in which X represents an integer from 0 to 10, Y represents hydrogen or a straight chain or branched alkyl radical of 1 to 5 carbon atoms which may be substituted by a carboxyl, hydroxyl or carbonamide group, the group ##EQU3## may also represent proline.
  • Y represents --CH 2 COOH or --CH 2 -CH 2 -COOH, and if X is an integer from 1 to 10, at least one lateral chain of Y is --CH 2 -COOH or --CH 2 -COOH.
  • W represents --OH, but in the presence of at least two carboxyl groups in the lateral chains, it may also represent --NH 2 , NH-C 2 H 5 , --OCH 3 or OC 2 H 5 .
  • the invention furthermore relates to a process for preparing the above-specified peptides, which comprises reacting the trypsin-callicrein inhibitor, whose 5 primary amino groups are protected by protective groups that can easily be split off with acids, with amino-acids or peptides of the general formula II ##EQU4## in the presence of 1-hydroxy-benzotriazole and dicyclohexylcarbodiimide and subsequently splitting off the protective groups with the aid of acids.
  • Y and X have the meanings given above, the carboxyl groups in Y, however, are present as tert.butyl esters.
  • W' represents tert.butoxy, but if at least 2 tert.butyl ester groups are present in the lateral chains, then W' may also represent --NH 2 , --NH-CH 3 , --NH-C 2 H 5 , --OCH 3 or OC 2 H 5 .
  • Boc-groups can be introduced with Boc-azide as well as with Boc-active esters, for example the p-nitrophenyl ester or the N-hydroxysuccinimide ester. In all cases, a product is formed which is uniform in paper electrophoresis.
  • N-protected compounds are reacted in the presence of 1-hydroxy-benzotriazole and dicyclohexylcarbodiimide with a compound of the general formula II in dimethylformamide or dimethylacetamide.
  • the protective groups are split off with trifluoroacetic acid or HCl/glacial acetic acid and the raw product is either dialyzed or chromatographed over Sephadex G 25.sup.(R), a cross-linked dextran gel.
  • the amino-acid rest or peptide or peptide group to be condensed must contain at least two carboxyl groups.
  • the naturally occurring amino-acids only aspartic acid and glutamic acid in their L- or D-form may be used.
  • dipeptides and higher peptides it is also possible to introduce, in addition to the acid amino-acids, other aliphatic amino-acids, for example glycine, alanine, leucine, valine, isoleucine, serine, threonine, asparagine, glutamine or proline in their L- or D-form.
  • the native inhibitor and modified preparations were subjected to micro-zone electrophoresis on cellulose-acetate foil in a diethyl-barbiturate buffer of pH 8.6.
  • the native inhibitor migrates in tthiis case towards the cathode, whereas the modified preparations migrate, depending on the modified group, more or less far towards the anode.
  • the migration distance towards the cathode is about 3 mm.
  • the migration distances towards the anode are about 1.5 mm, 8 mm, 13.5 mm and 17 mm respectively.
  • the modification of the inhibitor leads to preparations the basic character of which is reduced and the acid character of which is increased.
  • Modification with one group containing a single acid amino-acid already leads to such a lowering of the isoelectric point that at pH 8.6 the acid character slightly surpasses tthe basic character.
  • the isoelectric points estimated from the dissociation constants of the functional groups of the inhibitor and its derivatives prepared according to the invention are about 10.5 for the native inhibitor and about 8.1, 4.6, 4.35 and 4.1 for the modified preparations, the modified group containing one, two, three or four acid amino-acids.
  • the inhibitor solution was pre-incubated with a determined amount (corresponding to a determined activity) of trypsin solution; after a short time, a suitable trypsin substrate, for example N-benzoyl-arginine-4-nitroanilide (BAPA) was added and after some time and after having stopped the reaction, the yellow coloration due to p-nitroaniline released by uninhibited trypsin was measured quantitatively on a photometer at a suitable wave length, for example at 405 m ⁇ .
  • a suitable trypsin substrate for example N-benzoyl-arginine-4-nitroanilide (BAPA) was added and after some time and after having stopped the reaction, the yellow coloration due to p-nitroaniline released by uninhibited trypsin was measured quantitatively on a photometer at a suitable wave length, for example at 405 m ⁇ .
  • this process was carried out in that a quantitiy of trypsin (trypsin, crystallized, analytically pure) which, as previously determined, when combined within 30 minutes at 37° C with BAPA, causes a certain extinction at 405 m ⁇ , was incubated with increasing amounts of the inhibitor in a constant volume. After a pre-incubation of 30 minutes, the standard amount of BAPA was added. The reaction was stopped after 30 minutes at 37° C of BAPA incubation by the addition of dilute acetic acid and the yellow coloration was measured.
  • trypsin trypsin, crystallized, analytically pure
  • the trypsin-callicrein inhibitor modified according to the invention serves as a medicament in the treatment of hermorrhages caused by excessive fibrinolysis, for example in surgery in the case of prostate bleeding or disorders during the healing of wounds, in internal medicine as additional therapy in cases of hemophilia, in gynecology in cases of placenta praevia, fetal deatth in utero and atonic after-bleeding, and for prophylaxis in the case of operations of parenchymatous organs, as well as in cases of prostatectomies and fat embolisms.
  • the new medicaments of the invention are injected intravenously in sterile isotonic solution or administered as a slow drip infusion after dilution in infusion solutions, for example physiological salt solution. As doses, there may be administered 0.15 to 3 mg per kg.
  • Paper electrophoresis showed, in an acid medium, a uniform substance which was different from the trypsin-callicrein inhibitor.
  • the compound was prepared according to the methods described under (a), (b), (c) and (d) from the corresponding D-glutamic acid derivatives.
  • the melting points corresponded to those of the L-compounds.
  • the specific rotations of the D-compounds were also fouund to correspond in their values to those of the L-compounds, but had a reversed sign.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US05/503,066 1973-09-06 1974-09-04 Synthetically modified trypsin inhibitors and process for preparing them Expired - Lifetime US3953417A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US05/636,728 US3992529A (en) 1973-09-06 1975-12-01 Method of treating excessive fibrinolysis with synthetically modified trypsin inhibitors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19732344886 DE2344886C3 (de) 1973-09-06 Synthetisch modifizierte Derivate des basischen Trypsin-Kalllkrein-Inhibitors aus Säugetierorganen und Verfahren zu ihrer Herstellung
DT2344886 1973-09-06

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US05/636,728 Division US3992529A (en) 1973-09-06 1975-12-01 Method of treating excessive fibrinolysis with synthetically modified trypsin inhibitors

Publications (1)

Publication Number Publication Date
US3953417A true US3953417A (en) 1976-04-27

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US05/503,066 Expired - Lifetime US3953417A (en) 1973-09-06 1974-09-04 Synthetically modified trypsin inhibitors and process for preparing them

Country Status (12)

Country Link
US (1) US3953417A (fr)
JP (1) JPS5052081A (fr)
AT (1) AT339472B (fr)
BE (1) BE819639A (fr)
CA (1) CA1030524A (fr)
CH (1) CH608787A5 (fr)
EG (1) EG11583A (fr)
ES (1) ES429700A1 (fr)
FR (1) FR2242992B1 (fr)
GB (1) GB1462493A (fr)
NL (1) NL7411563A (fr)
SE (1) SE414172B (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3992529A (en) * 1973-09-06 1976-11-16 Hoechst Aktiengesellschaft Method of treating excessive fibrinolysis with synthetically modified trypsin inhibitors
WO2007099650A1 (fr) * 2006-03-01 2007-09-07 Fukuoka Prefectural Government Vecteur contenant un lipide peptidique et procede permettant d'introduire un compose dans des cellules l'utilisant
US20100297120A1 (en) * 2007-05-29 2010-11-25 Angiochem Inc. Aprotinin-like polypeptides for delivering agents conjugated thereto to tissues
US10980892B2 (en) 2015-06-15 2021-04-20 Angiochem Inc. Methods for the treatment of leptomeningeal carcinomatosis

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3135541A1 (de) * 1981-09-08 1983-03-24 Bayer Ag, 5090 Leverkusen Kallikrein-trypsin-inhibitor-(bpti)-derivate, traegergebundene bpti-derivate, ihre herstellung und ihre verwendung zur herstellung der reinen enzyme trypsin, chymotrypsin und kallikrein

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3558773A (en) * 1966-02-23 1971-01-26 Bayer Ag Crystalline kallikrein-inactivator and process for preparing the same
US3798205A (en) * 1967-07-26 1974-03-19 Bayer Ag Derivatives of the kallikrein inhibitor and their production
US3847893A (en) * 1972-03-01 1974-11-12 Bayer Ag Process for the preparation of insulin derivatives cross-linked by a dicarboxylic acid group at the amino a-1 glycine and amino b-29 lysine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3558773A (en) * 1966-02-23 1971-01-26 Bayer Ag Crystalline kallikrein-inactivator and process for preparing the same
US3798205A (en) * 1967-07-26 1974-03-19 Bayer Ag Derivatives of the kallikrein inhibitor and their production
US3847893A (en) * 1972-03-01 1974-11-12 Bayer Ag Process for the preparation of insulin derivatives cross-linked by a dicarboxylic acid group at the amino a-1 glycine and amino b-29 lysine

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3992529A (en) * 1973-09-06 1976-11-16 Hoechst Aktiengesellschaft Method of treating excessive fibrinolysis with synthetically modified trypsin inhibitors
WO2007099650A1 (fr) * 2006-03-01 2007-09-07 Fukuoka Prefectural Government Vecteur contenant un lipide peptidique et procede permettant d'introduire un compose dans des cellules l'utilisant
US20090170960A1 (en) * 2006-03-01 2009-07-02 Fukuoka Prefectural Government Peptide lipid-containing carrier and method for introducing compound into cells using same
US8299129B2 (en) 2006-03-01 2012-10-30 Fukuoka Prefectural Government Peptide lipid-containing carrier and method for introducing compound into cells using same
US20100297120A1 (en) * 2007-05-29 2010-11-25 Angiochem Inc. Aprotinin-like polypeptides for delivering agents conjugated thereto to tissues
US9365634B2 (en) 2007-05-29 2016-06-14 Angiochem Inc. Aprotinin-like polypeptides for delivering agents conjugated thereto to tissues
US10980892B2 (en) 2015-06-15 2021-04-20 Angiochem Inc. Methods for the treatment of leptomeningeal carcinomatosis

Also Published As

Publication number Publication date
NL7411563A (nl) 1975-03-10
SE414172B (sv) 1980-07-14
AT339472B (de) 1977-10-25
DE2344886A1 (de) 1975-04-03
DE2344886B2 (de) 1976-08-12
CA1030524A (fr) 1978-05-02
FR2242992A1 (fr) 1975-04-04
ATA716374A (de) 1977-02-15
EG11583A (en) 1978-03-29
FR2242992B1 (fr) 1980-01-11
AU7296374A (en) 1976-03-11
BE819639A (fr) 1975-03-06
ES429700A1 (es) 1977-03-01
GB1462493A (en) 1977-01-26
CH608787A5 (fr) 1979-01-31
JPS5052081A (fr) 1975-05-09
SE7411157L (fr) 1975-03-07

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